This proposal describes the development of a high throughput screening assay for small molecules effective against Human Immunodeficiency Virus Type 1 (HIV-1) fusion. Preventing HIV-1 fusion would inhibit the entry of virus into human host cells, effectively protecting uninfected cells and improving the available treatment options for HIV infected patients. Currently there is a single fusion inhibitor, a peptide Enfuvirtide(r), which is expensive and available only by intravenous administration. Drugs which can be taken orally are low molecular weight compounds which usually can be manufactured at lower cost and be more widely distributed. The screening assay will be capable of automated testing of thousands of low molecular weight compounds available in academic, government and pharmaceutical facilities. Compounds selected by the initial screen would be subject to further testing and modification to improve potency. The assay described involves the simple addition of two peptides derived from HIV-1 gp41, the viral fusion protein, to wells of plated library compounds. The peptides are modified with a fluorophore and metal-ligated dye complex, which enables their micromolar association to be followed by a simple fluorescence intensity readout. Compounds from a library are assessed for activity by their ability to competitively inhibit the peptide association, with a concomitant fluorescence increase. A positive result indicates that a compound is capable of fusion inhibition. The intensity of the fluorescence signal is directly correlated to the compound's potency. This is a biochemical assay, using inexpensive and non-hazardous components. We will show that it is highly specific for the viral target, extremely robust and sensitive, and an excellent indicator of a compound's ability to inhibit fusion in cell culture. In this study, we will optimize the assay for maximum sensitivity, broaden the selection of small molecules to include fragments that could be tethered to create more powerful inhibitors, and explore NMR and fluorescence methods for facilitating the optimization process of newly discovered hits from a library. This project will enable systematic screening of compound libraries for HIV-1 fusion inhibitors. The screen will specifically identify molecules that bind to the gp41 fusion protein in such as way as to prevent the conformational change required for effective viral fusion. Newly discovered small molecule candidates may be developed into entry inhibitors effective in controlling HIV-1 infection, as part of a multi-drug treatment strategy. [unreadable] [unreadable] [unreadable]